Phylogenomic reappraisal of the family Rhizobiaceae at the genus and species levels, including the description of Ectorhizobium quercum gen. nov., sp. nov.

The family Rhizobiaceae contains 19 validly described genera including the rhizobia groups, many of which are important nitrogen-fixing bacteria. Early classification of Rhizobiaceae relied heavily on the poorly resolved 16S rRNA genes and resulted in several taxonomic conflicts. Although several recent studies illustrated the taxonomic status of many members in the family Rhizobiaceae, several para- and polyphyletic genera still needed to be elucidated. The rapidly increasing number of genomes in Rhizobiaceae has allowed for a revision of the taxonomic identities of members in Rhizobiaceae. In this study, we performed analyses of genome-based phylogeny and phylogenomic metrics to review the relationships of 155-type strains within the family Rhizobiaceae. The UBCG and concatenated protein phylogenetic trees, constructed based on 92 core genes and concatenated alignment of 170 single-copy orthologous proteins, demonstrated that the taxonomic inconsistencies should be assigned to eight novel genera, and 22 species should be recombined. All these reclassifications were also confirmed by pairwise cpAAI values, which separated genera within the family Rhizobiaceae with a demarcation threshold of ~86%. In addition, along with the phenotypic and chemotaxonomic analyses, a novel strain BDR2-2T belonging to a novel genus of the family Rhizobiaceae was also confirmed, for which the name Ectorhizobium quercum gen. nov., sp. nov. was proposed. The type strain is BDR2-2T (=CFCC 16492T = LMG 31717T).

The early taxonomic classification of Rhizobiaceae was circumscribed by two genera, namely Rhizobium and Agrobacterium, only based on nitrogen-fixing or pathogenic traits. As the phylogenetic analysis advances, many other novel genera belonging to the family Rhizobiaceae were proposed in succession based on the 16S rRNA phylogenetic trees [e.g., Martelella (Rivas et al., 2005), Shinella (An et al., 2006), Ciceribacter (Kathiravan et al., 2013), Lentilitoribacter (Park et al., 2013), Liberibacter (Fagen et al., 2014), and Gellertiella (Tóth et al., 2017)]. Over time, the phylogenetic method of multilocus sequence analysis (MLSA), which provides a more robust taxonomic resolution (De Lajudie et al., 2019), was used in the revision of the genera within Rhizobiaceae, and the proposal of several novel genera and combinations made the classifications of Rhizobiaceae more precise (Ramírez-Bahena et al., 2008;Kimes et al., 2015;Rocha et al., 2020). At present, the defined prokaryotic genera or higher taxa relies heavily on the monophyly of species (De Lajudie et al., 2019), and the genome-based phylogeny was considered to be a more convenient and accurate method (Parks et al., 2018). Benefiting from the advances in next-generation sequencing technology, an enormous amount of genomic data had been accumulated in public databases, which provided the base for a more accurate classification of prokaryotes. Based on the genomic data of the family Rhizobiaceae in the public databases, the genus Ciceribacter , Agrobacterium tumefaciens species complex G3 (Singh et al., 2021), and Rhizobium leguminosarum species complex (Young et al., 2021) were revised, along with various genomic metrics, and several new species combinations were proposed. Recently, a genomic metric of cpAAI data was proposed to define the genera in the family Rhizobiaceae with a threshold of ∼86%, and several new genera and combinations were proposed (Kuzmanović et al., 2022). However, there are several taxonomic inconsistencies within Rhizobiaceae that need to be elucidated.
During our study of the bacterial diversity in the disease of oaks, the strain BDR2-2 T was isolated from the symptomatic bark of Quercus acutissima caker. Preliminary phylogeny analysis showed that the strain BDR2-2 T should be assigned to the family Rhizobiaceae. In this study, we combined the UBCG and 120 ubiquitous single-copy protein phylogenetic analyses, along with the genomic metrics of AAI, POCP, and cpAAI, to confirm the taxonomic status of BDR2-2 T and other conflicts of species within the family Rhizobiaceae.
. Materials and methods

. . Strain and culture conditions
The strain BDR2-2 T was isolated from the symptomatic bark of Q. acutissima caker collected from Hefei, China (31 • 50 ′ 28 ′′ N, 117 • 10 ′ 34 ′′ E). The isolation and purification of the strain BDR2-2 T were performed as previously described (Ma et al., 2022). In brief, the samples were initially surface-sterilized successively in 70% ethanol for 30 s and 4% (v/v) sodium hypochlorite for 2 min. After washing with sterile water for three times, the samples were transferred to a sterile mortar, ground with a pestle, and then cultivated for 30 min. The suspensions were spread on yeast extract mannitol agar (YMA) with a dilution series. After 2 days of incubation at 30 • C, single colonies were cultured on a new plate and then preserved at −80 • C.

. . Genome sequencing and reference genome
The genome of the strain BDR2-2 T was sequenced with Illumina NovaSeq PE150 by Novogene, Co., Ltd. (Beijing, China). In brief, the low-quality reads were filtered by readfq (version 10), and then, the high-quality reads were assembled using SOAPdenovo (version 2.04) (Li et al., 2008(Li et al., , 2010, SPAdes (Bankevich et al., 2012), and ABySS (Simpson et al., 2009). After integrating with CISA (Lin and Liao, 2013), the gaps in the results were filled with gapclose (version 1.12). In this study, 136 validated Rhizobiaceae and 18 unvalidated Rhizobiaceae were analyzed. Because the 16S rRNA phylogenetic tree was inappropriate for delineating genera in the family Rhizobiaceae in previous studies (e.g., strains from Brucellaceae nested in Rhizobiaceae) (Hördt et al., 2020), five type strains from Brucellaceae (including the type genus Brucella and type species from the other two genera) were also analyzed in this study to confirm it. In addition, five strains from Caulobacterales were also used as an out-group in this study. The type strain genome sequences used were obtained from the NCBI database, and all of the genome sequences were assessed by CheckM (Parks et al., 2015).

. . Phylogenetic analyses
Full-length 16S rRNA gene sequences were extracted from the genomes via RNAmmer 1.2 for the phylogenetic analysis (Lagesen et al., 2007). The multiple alignments of the sequences were performed with Clustal W, and then, the phylogenetic trees were constructed with MEGA X by the methods of maximum-likelihood, neighbor-joining, and maximum-parsimony (Kumar et al., 2018). The phylogenetic trees were evaluated by 1,000 bootstrap resamplings, and the species of Brucellaceae and Caulobacterales were used as the out-group.
A phylogenomic tree, particularly a concatenated core gene tree, was considered to be a more convenient and accurate substitute method for taxonomic analysis as it provides a higher resolution phylogeny (Kim et al., 2021). There are 92 core genes that were extracted from the genomes using the command "java -jar UBCG.jar extract" and used in the UBCG phylogenetic tree, which was generated by RAxML using the command "jar -jar UBCG.jar align." The species of Brucellaceae and Caulobacterales were used as the out-group.
Additionally, another phylogenomic tree with a concatenated alignment of 170 ubiquitous single-copy proteins was constructed with FastTree. The extraction and alignment of the sequences were generated with the method at github.com/flass/cpAAI_Rhizobiaceae (Kuzmanović et al., 2022), and the tree was visualized and edited with iTOL (Letunic and Bork, 2021).

. . Chemotaxonomy and physiology
The polar lipids and isoprenoid quinones were performed as described by Minnikin et al. (1984) and Collins et al. (1977), respectively. The extraction of cellular fatty acids was performed as described by Kuykendall et al. (1988) and then analyzed with the Sherlock Microbial Identification System (MIDI) (Sasser, 1990). The growth gradients of pH, temperature, and salinity were optimized by the methods described by Li et al. (2016). Gram staining was carried out as described by Jenkins et al. (2003). The test of anaerobic growth was performed in an anaerobic jar for a week (Li et al., 2016). The activities of oxidase and catalase were determined by the methods described by Li et al. (2016). Enzymatic activity, acid production, and carbon source utilization were performed using API ZYM, API 50CH, and API 20NE (bioMérieux) according to the manufacturer's instructions.

. Results and discussion
The 16S rRNA gene phylogeny was widely used in prokaryote taxonomic analyses due to its high conservation (Park et al., 2013;Fagen et al., 2014;Tóth et al., 2017), and this, on the other hand, generally did not provide sufficient resolution for closely related species (Vinuesa et al., 2005;Liang et al., 2021). As expected, the full-length 16S rRNA phylogenetic tree showed low bootstrap support at the genus and species levels, resulting in poorly resolved taxonomic issues (Supplementary Figure S1). The concatenated proteins and UBCG trees showed a similar phylogenetic backbone to each other, and most of the species in the family Rhizobiaceae consistently grouped into similar monophyletic clades with high bootstrap values. The concatenated protein is shown in Figure 1, and the full details of the two phylogenetic trees are shown in Supplementary Figures S2, S3. For genus demarcation within Rhizobiaceae, the genomic metric of cpAAI data was recently proposed, with a threshold of ∼86% (Kuzmanović et al., 2022), and here we calculated the pairwise cpAAI values to confirm the reclassification of the Rhizobiaceae order.
As shown in the pairwise AAI, POCP, and cpAAI values of the currently proposed genera in the family Rhizobiaceae, the pairwise values between inter-genus and intra-genus could not be separated (Figures 2A, C), and there should be several misclassification species among the currently proposed genera. By applying the cpAAI threshold of ∼86% for genus demarcation and combining it with phylogenetic tree analysis, most of the pairwise values could be clearly separated between inter-genus and intra-genus ( Figures 2B, D). The pairwise AAI, POCP, and cpAAI values are shown in Supplementary Table S1.
. . Reclassification of Rhizobiaceae at the genus level showed that most genera within the family Rhizobiaceae were clustered into monophyletic clades, except for several genera that formed paraphyletic or polyphyletic clades. Among those paraphyletic or polyphyletic clades, the taxonomic conflicts were resolved as follows. All reclassifications were also confirmed by the genus demarcation of cpAAI with a threshold of ∼86%.
As for the paraphyletic genus, which consisted of a monophyletic clade with one or more species of a different genus (Wood, 1994;Liang et al., 2021), the conflicting clade should be merged into the primary genus. Martelella appeared like a paraphyletic genus in both phylogenetic trees because Martelella alba BGMRC 2036 T , a recently proposed novel species, formed an outermost clade of the Martelella lingage by a long branch, and M. alba BGMRC 2036 T might be a different genus from Martelella. Furthermore, the pairwise cpAAI values between M. alba BGMRC 2036 T and other Martelella strains ranged from 79.5 to 80.5% (Supplementary Table S1 and Supplementary Figure S4A), which were also significantly lower than the recommended genus demarcation value of 86%, and therefore, we proposed to transfer M. alba BGMRC 2036 T to a novel genus Paramartelella gen. nov.
Mycoplana was shown as paraphyletic in all phylogenetic trees ( Figure 1, Supplementary Figures S2, S3) because Rhizobium rhizolycopersici DBTS2 T was nested within Mycoplana with high support. In the original proposal of R. rhizolycopersici DBTS2 T , the phylogenetic tree was constructed with a low number of closely related taxa, and the Mycoplana-type strain was not considered (Thin et al., 2021). Therefore, we proposed to assign R. rhizolycopersici DBTS2 T to Mycoplana. In addition, the pairwise cpAAI values within the Mycoplana clade ranged from 89.7 to 97.2% (Supplementary Table S1 and Supplementary Figure S4B), and those values were also higher than the genus demarcation threshold, which further confirmed the classification.
Similarly, the genera Peteryoungia, Agrobacterium, Neorhizobium also appeared as paraphyletic in all phylogenetic trees ( Figure 1, Supplementary Figures S2, S3). Rhizobium glycinendophyticum CL12 T and Agrobacterium albertimagni AOL15 T were nested within Peteryoungia with a high bootstrap value. The pairwise cpAAI values between the two strains and other Peteryoungia strains were also higher than   the genus demarcation threshold (Supplementary Table S1 and Supplementary Figure S4C), which confirmed that R. glycinendophyticum CL12 T and A. albertimagni AOL15 T should be transferred to Peteryoungia. Using a similar method as above, Rhizobium oryzihabitans M15 T was nested within Agrobacterium, and Rhizobium deserti SPY 1 T , Rhizobium populisoli XQZ8 T , and Rhizobium terrae NAU 18 T were nested within Neorhizobium.
Pairwise cpAAI values confirmed that R. oryzihabitans M15 T should be assigned to Agrobacterium (Supplementary Table S1 and Supplementary Figure S4D), and R. deserti SPY 1 T , R. populisoli XQZ8 T , and R. terrae NAU 18 T should be assigned to Neorhizobium (Supplementary Table S1 and Supplementary Figure S4E). Different from the paraphyletic genus, the polyphyletic genus was typically more difficult to resolve, as the taxonomic issues were done by merging the conflicting clades or transferring them to novel genera (Farris, 1974;Liang et al., 2021). Pararhizobium .
/fmicb. . appeared as polyphyletic in the phylogenetic trees ( Figure 1, Supplementary Figures S2, S3) because Pararhizobium mangrovi BGMRC 6574 T and Pararhizobium haloflavum XC0140 T were placed in a distant position relative to the genus Pararhizobium with high support values. This analysis confirmed P. haloflavum XC0140 T , which was not validly published despite being proposed as "Neopararhizobium" (Hördt et al., 2020) and represents a novel genus. Pararhizobium mangrovi BGMRC 6574 T , a recently proposed novel species, formed the outermost clade of "Neopararhizobium" and Georhizobium lineages with a distant evolutionary relationship with Pararhizobium in all phylogenetic trees, which implied that the strain should be assigned to a novel genus. The pairwise cpAAI values between P. mangrovi BGMRC 6574 T and other Pararhizobium strains ranged from 69.4 to 69.5%, which were also significantly lower than the recommended genus demarcation value of 86%, and therefore, we proposed to transfer P. mangrovi BGMRC 6574 T to a novel genus Allopararhizobium gen. nov. Although most taxonomic conflicts of the genus Rhizobium were resolved, Rhizobium was shown as polyphyletic in the phylogenetic trees ( Figure 1, Supplementary Figures S2, S3), including strains such as Rhizobium album NS-104 T , Rhizobium halophytocola DSM 21600 T , Rhizobium clade 2, Rhizobium populi CCTCC AB 2013068 T , Rhizobium clade 3, and Rhizobium clade 4, which were placed apart from the genus Rhizobium. Rhizobium album NS104 T formed the outermost clade of the genus Rhizobium lineage in all phylogenetic trees and showed a distant evolutionary relationship with other Rhizobium species, indicating that the sole species might represent a novel genus in the family Rhizobiaceae. In addition, the pairwise cpAAI values between R. album NS104 T and other Rhizobium strains ranged from 80.6 to 82.3% (Supplementary Table S1 and Supplementary Figure S4F), which were also significantly lower than the recommended genus demarcation value, and therefore R. album NS104 T represented a novel genus Metarhizobium gen. nov.
Rhizobium halophytocola DSM 21600 T , Rhizobium clade 2, Rhizobium clade 3, and Rhizobium clade 4, which formed four different highly supported monophyletic clades, were placed apart from the Rhizobium lineage in the phylogenetic trees and separated from the species in the genus Rhizobium, implying that they should be transferred to four different novel genera. With the similar analytical methods as above, the pairwise cpAAI values also confirmed that R. halophytocola DSM 21600 T and Rhizobium clades 2-4 should belong to four different novel genera in the family Rhizobiaceae (Supplementary Table S1 and Supplementary Figures S5G-J). The pairwise cpAAI values within the Rhizobium lineage were also significantly higher than these values between R. album NS104 T , R. halophytocola DSM 21600 T , Rhizobium clades 2-4, and Rhizobium lineage (Figure 3), which also confirmed that these clades belong to five different novel genera.
Altogether, R. halophytocola DSM 21600 T should be assigned to a novel genus Heterorhizobium gen. nov. Rhizobium clade 2 should belong to a novel genus Paenirhizobium gen. nov., with Paenirhizobium daejeonense comb. nov. as the type species. Rhizobium clade 3 should be assigned to Affinirhizobium gen. nov., with Affinirhizobium pseudoryzae comb. nov. as the type species. Rhizobium clade 4 should be assigned to Alirhizobium gen. nov., with Alirhizobium cellulosilyticum comb. nov. as the type species.

. . . Genome-based phylogenetic analyses
The 16S rRNA sequence pairwise comparisons showed that the strain BDR2-2 T was most closely related to Allorhizobium borbori DN316 T (97.4% similarity), followed by R. populi K-38 T (96.9% similarity), and less than 96.5% similarity with other species of the family Rhizobiaceae in the EzBioCloud database (Yoon et al., 2017), and therefore, the strain BDR2-2 T might belong to a novel species of Rhizobiaceae. The strain BDR2-2 T , A. borbori DN316 T , and R. populi K-38 T consistently formed a highly supported monophyletic lineage closer to Gellertiella hungarica DSM 29853 T or other lineages than to the genus Allorhizobium and Rhizobium lineages (Figures 1, 4), indicating that the three strains should be allocated to novel Rhizobiaceae genera. The cpAAI values between BDR2-2 T and A. borbori DN316 T were significantly higher than the genus demarcation threshold, indicating that Allorhizobium clade 2 should belong to the same genus. The pairwise cpAAI values between R. populi K-38 T and Allorhizobium clade 2 were 84.9 and 85.3%, respectively (Figure 4), which were slightly lower than the genus demarcation threshold. While the 86% threshold was an approximation and not strictly unique, and species within the same genus might have different evolutionary rates (Ramette and Tiedje, 2007;Liang et al., 2021), therefore, we proposed to tentatively place R. populi K-38 T in Allorhizobium clade 2 and assign the three strains to a novel genus. In addition, the chemotaxonomic and physiological analyses revealed that the three strains shared similar major phenotypic features with each other (Table 1 and Supplementary Table S2), which also confirmed the reclassification.
The ANI and dDDH values, which are gold standards for species delineation (Liang et al., 2021), were lower in

. Conclusion
Since the low resolution of 16S rRNA phylogeny on the closely related species was an important cause of the taxonomic issue, we, therefore, constructed two genome-based phylogenetic trees, namely concatenated proteins tree and UBCG tree, to resolve the misclassifications. Genome sequences from 138 of the 181 validly published Rhizobiaceae species, 18 not validly published Rhizobiaceae species were used to confirm the taxonomic status of species in the family Rhizobiaceae, five Brucellaceae and five Caulobacterales were used as the out-group. Along with the phylogenomic metric analyses of cpAAI, eight novel genera, one novel species, and 22 novel combinations were proposed. Cells are Gram-strain-negative, motile, aerobic, and rodshaped. The predominant respiratory quinone is Q-10. The major cellular fatty acids usually contain C 19 : 0 cyclo ω8c. The DNA G+C .
/fmicb. . content is 64.7 mol%. Species of the genus are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type species is Allopararhizobium mangrovi comb. nov.
Cells are Gram-strain-negative, non-motile, catalase-positive, and rod-shaped. The predominant respiratory quinone is Q-10. The major cellular fatty acids usually contain summed feature 8 (comprising C 18 : 1 ω7c and/or C 18 : 1 ω6c). The DNA G+C content is 62.3 mol%. Species of the genus are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type species is Paramartelella alba comb. nov.
Cells are Gram-strain-negative, motile, facultatively anaerobic, catalase-and oxidase-positive, and rod-shaped. The predominant respiratory quinone is Q-10. The major cellular fatty acids usually contain C 19 : 0 cyclo ω8c and C 18 : 1 ω7c. The DNA G+C content is 61.9 mol%. Species of the genus are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type species is Metarhizobium album comb. nov.
Cells are Gram-strain-negative, motile, catalase-and oxidasepositive, aerobic, and rod-shaped. The respiratory quinone is Q-10. The major cellular fatty acids usually contain C 18 : 1 ω7c and C 19 : 0 cyclo ω8c. The DNA G+C content is 52.8 mol%. Species of the genus are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type species is Heterorhizobium halophytocola comb. nov.
Cells are Gram-stain-negative, aerobic, catalase-, and oxidasepositive. The predominant respiratory quinone is Q-10. The major cellular fatty acids usually contain summed feature 8 (comprising C 18 : 1 ω7c and/or C 18 : 1 ω6c) and C 16 : 0. The DNA G+C content is 61.3-64.5 mol%. Members of the genus are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type species is Ectorhizobium quercum sp. nov.
Cells are Gram-strain-negative, catalase-positive, aerobic, and rod-shaped. The DNA G + C content is 59.3-60.2 mol%. The major cellular fatty acids usually contain summed feature 8 (comprising C 18 : 1 ω7c and/or C 18 : 1 ω6c). Species of the genus are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type species is Affinirhizobium pseudoryzae comb. nov.
Basonym: Pararhizobium mangrovi Li et al., 2021. The description of A. mangrovi is the same as that given for P. mangrovi (Li et al., 2021b). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is BGMRC 6574 T (= CGMCC 1.16783 T = KCTC 72636 T ).
Paramartelella alba (al'ba. L. fem. adj. alba, white, referring to the color of the colonies).
Basonym: Martelella alba Li et al., 2021. The description of P. alba is the same as that given for M. alba (Li et al., 2021a). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is BGMRC 2036 T (= KCTC 52121 T = NBRC 111908 T ).
Basonym: Rhizobium rhizolycopersici Thin et al., 2021. The description of M. rhizolycopersici is the same as that given for R. rhizolycopersici (Thin et al., 2021). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is DBTS2 T (= CICC 24887 T = ACCC61707 T = JCM 34245 T ). The description of H. halophytocola is the same as that given for R. halophytocola (Bibi et al., 2012). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is YC6881 T (= DSM 21600 T = KACC 13775 T ).
Basonym: Rhizobium naphthalenivorans Kaiya et al., 2018. The description of Paenirhizobium naphthalenivorans is the same as that given for Rhizobium naphthalenivorans (Kaiya et al., 2012). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is TSY03b T (= KCTC 23252 T = NBRC 107585 T ).
Basonym: Rhizobium selenitireducens Hunter et al., 2008. The description of Paenirhizobium selenitireducens is the same as that given for Rhizobium selenitireducens (Hunter et al., 2007). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is B1 T (= NRRL B-41997 T = LMG 24075 T = ATCC BAA-1503 T ).
Basonym: Rhizobium glycinendophyticum Wang et al., 2020. The description of P. glycinendophyticum is the same as that given for R. glycinendophyticum . The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is CL12 T (= KACC 21281 T = GDMCC 1.1597 T ).
Peteryoungia albertimagni (albertimagni, is named after the Dominican scholar Albertus Magnus, who was the first person to describe arsenic).
The description of P. albertimagni is the same as that given for A. albertimagni (Salmassi et al., 2002). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is AOL15 T = ATCC BAA-24 T ).
. . . Description of Ectorhizobium borbori comb.nov. The description of Ectorhizobium borbori is the same as that given for Rhizobium borbori (Zhang G. X. et al., 2011). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is DN316 T (= CICC 10378 T = LMG 23925 T = DSM 22790 T =DSM 26385 T =HAMBI 3454 T ).
Ectorhizobium populi (po'pu.li. L. gen. fem. n. populi, of a poplar tree, pertaining to Populus euphratica, the Latin name for the poplars that grow in the forest from which the type strain was isolated).
Basonym: Rhizobium populi Rozahon et al., 2014. The description of E. populi is the same as that given for R. populi (Rozahon et al., 2014). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is K-38 T (= CCTCC AB 2013068 T = NRRL B-59990 T = JCM 19159 T ).
Basonym: Rhizobium oryzihabitans Zhao et al., 2020. The description of A. oryzihabitans is the same as that given for R. oryzihabitans (Zhao et al., 2020). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is M15 T (= JCM 32903 T = ACCC 60121 T ). The description of Affinirhizobium pseudoryzae is the same as that given for Rhizobium pseudoryzae . The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is J3-A127 T (= ACCC 10380 T = KCTC 23294 T = DSM 19479 T = DSM 26483 T ).
The description of Affinirhizobium helianthi is the same as that given for Rhizobium helianthi (Wei et al., 2015). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is Xi19 T (= CGMCC 1.12192 T = KCTC 23879 T ).
Basonym: Rhizobium rhizoryzae . The description of Affinirhizobium rhizoryzae is the same as that given for Rhizobium rhizoryzae . The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is J3-AN59 T (= ACCC 05916 T = DSM 19478 T = DSM 29514 T = KCTC 23652 T ).
The description of Alirhizobium cellulosilyticum is the same as that given for Rhizobium cellulosilyticum (Garcia-Fraile et al., 2007). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is ALA10B2 T (=DSM 18291 T = CECT 7176 T = LMG 23642 T ).
Alirhizobium wenxiniae (wen.xin'i.ae. N.L. gen. fem. n. wenxiniae, of Wen-xin, to honor Wen-xin Chen, a respected rhizobial taxonomist, for her great contributions to the investigation and taxonomy of rhizobial resources in China).
Basonym: Rhizobium wenxiniae Gao et al., 2017. The description of Alirhizobium wenxiniae is the same as that given for Rhizobium wenxiniae (Gao et al., 2017). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is 166 T (= DSM 100734 T = CGMCC 1.15279 T ).
The description of Alirhizobium smilacinae is the same as that given for Rhizobium smilacinae . The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is PTYR-5 T (= DSM 100675 T = CCTCC AB 2013016 T = KCTC 32300 T = LMG 27604 T ).
Basonym: Rhizobium deserti Liu et al., 2020. The description of N. deserti is the same as that given for R. deserti (Liu et al., 2020). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is SPY-1 T (= ACCC 61627 T = JCM 33732 T ).
Neorhizobium terrae (ter'rae. L. gen. fem. n. terrae, of soil, referring to the isolation source of the type strain).
Basonym: Rhizobium terrae Ruan et al., 2021. The description of N. terrae is the same as that given for R. terrae (Ruan et al., 2020). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is NAU-18 T (= CCTCC AB 2018075 T = KCTC 62418 T ).
Neorhizobium populisoli (po.pu.li.so'li. L. fem. n. Populus, the poplar tree (genus Populus); L. neut. adj. solum, soil; N.L. gen. neut. n. populisoli, of poplar soil, referring to the isolation of the bacterium from the rhizosphere soil of P. popularis). The description of N. populisoli is the same as that given for R. populisoli (Shen et al., 2022). The species are classified based on UBCG and concatenated protein phylogenetic trees, as well as phylogenomic metric analyses of cpAAI. The type strain is XQZ8 T (=JCM 34442 T = GDMCC 1.2201 T ).

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: NCBI-JANFPI010000000 and PRJNA859997.

Author contributions
CP and YL designed the experiment, provided the methods, and revised the manuscript. TM finished the manuscript and completed most of the experiments. HX and NJ helped to reconstruct and analyze the gene trees. All authors read and approved the final version of the manuscript.